GSK2801 Reverses Paclitaxel Resistance in Anaplastic Thyroid Cancer Cell Lines through MYCN Downregulation

Anaplastic thyroid cancer (ATC) is a very rare, but extremely aggressive form of thyroid malignancy, responsible for the highest mortality rate registered for thyroid cancer. Treatment with taxanes (such as paclitaxel) is an important approach in counteracting ATC or slowing its progression in tumors without known genetic aberrations or those which are unresponsive to other treatments. Unfortunately, resistance often develops and, for this reason, new therapies that overcome taxane resistance are needed. In this study, effects of inhibition of several bromodomain proteins in paclitaxel-resistant ATC cell lines were investigated. GSK2801, a specific inhibitor of BAZ2A, BAZ2B and BRD9, was effective in resensitizing cells to paclitaxel. In fact, when used in combination with paclitaxel, it was able to reduce cell viability, block the ability to form colonies in an anchor-independent manner, and strongly decrease cell motility. After RNA-seq following treatment with GSK2801, we focused our attention on MYCN. Based on the hypothesis that MYCN was a major downstream player in the biological effects of GSK2801, we tested a specific inhibitor, VPC-70619, which showed effective biological effects when used in association with paclitaxel. This suggests that the functional deficiency of MYCN determines a partial resensitization of the cells examined and, ultimately, that a substantial part of the effect of GSK2801 results from inhibition of MYCN expression.


Introduction
Thyroid cancer (TC) is the most common endocrine carcinoma, accounting for 1-2% of cancer cases worldwide [1]. Thyroid cancer originates from follicular epithelial cells or parafollicular C cells. The ones derived from the follicular cells are classified into four histological types: papillary thyroid cancer (PTC, 80-85%), follicular thyroid cancer (FTC, 10-15%), poorly-differentiated thyroid cancer (PDTC, <2%), and anaplastic thyroid cancer (ATC, <2%) [2]. PTC and FTC account for more than 95% of tumors and are mostly responsive to current treatment based on surgery and subsequent radioiodine therapy, with an overall survival rate of more than 90% within 10 years. The situation is different regarding ATC, which is a very rare but extremely aggressive form of thyroid neoplasm. In fact, although it accounts for a very small percentage of all thyroid malignancies (less than 2%), it is mostly diagnosed as stage IV disease, is often unresectable, and fails to respond to radioactive iodine ablation because of its undifferentiated phenotype due to loss of thyroid-specific gene expression. Therefore, ATC is responsible for 20-50% of thyroid cancer mortality [3]. Stage IVA and IVB (when resectable) ATCs are treated by surgical resection followed by intensity-modulated radiation therapy (IMRT) and taxane-based chemotherapy alone or in combination with platinum or anthracycline [4]. For not-resectable stage IVB ATC, two classes of FDA-approved drugs are presently available: tyrosine kinase inhibitors (TKIs) and mutation-specific drugs [5]. However, in some patients, both of these strategies fail [6].

Results
Since in a previous study, mRNA-seq analysis had shown expression of GSK2801 targets (BAZ2A, BAZ2B and BRD9) in SW1736 and 8505C paclitaxel-resistant cells [3], we wondered whether these bromodomain protein actions represent a mechanism of resistance to taxane treatment in this model. Figure 1 shows that in paclitaxel-resistant ATC cells (8505C-PTX and SW1736-PTX), the expression level of BRD9 is increased compared with non-resistant parental lines both at the mRNA and protein levels (Figure 1, panel A and C). In contrast, gene expression analysis and Western blot analysis of BAZ2A and BAZ2B show no significant change in resistant cells. the ChIP-seq approach, it was recently discovered that GSK2801 significantly disrupts chromatin binding of flag-tagged BAZ2A or BAZ2B in mouse liver H2.35 cells [27].

Results
Since in a previous study, mRNA-seq analysis had shown expression of GSK2801 targets (BAZ2A, BAZ2B and BRD9) in SW1736 and 8505C paclitaxel-resistant cells [3], we wondered whether these bromodomain protein actions represent a mechanism of resistance to taxane treatment in this model. Figure 1 shows that in paclitaxel-resistant ATC cells (8505C-PTX and SW1736-PTX), the expression level of BRD9 is increased compared with non-resistant parental lines both at the mRNA and protein levels (Figure 1, panel A and C). In contrast, gene expression analysis and Western blot analysis of BAZ2A and BAZ2B show no significant change in resistant cells.  protein levels in these four cell lines. For each cell line, the results were normalized against β-actin levels and expressed in arbitrary unit. Protein expression in the paclitaxel-sensitive parental cell line was set at 1 and data are expressed as fold change respect to control. n = 3, * p < 0.05, ** p < 0.01.
In this study, the biological effects of the BAZ2A, BAZ2B, and BRD9 specific inhibitor GSK2801 on two resistant ATC cell lines were investigated. In a first experimental setting, we evaluated the effect on cell viability of different doses of GSK2801 in two paclitaxelsensitive ATC cell lines (SW1736 and 8505C, Figure 2 panel A) and in two paclitaxel-resistant ATC cell lines (SW1736-PTX and 8505C-PTX to GSK2801, Figure 2, panel B), alone or in combination with paclitaxel ( Figure 2, panel C). Treatments with GSK2801 show very modest effects in paclitaxel-sensitive cells or in SW1736-PTX and 8505C-PTX cell lines; moreover, there is no dose-response effect as even when increasing the dose up to 40 µM, no effect is shown in SW1736, 8505C, and SW1736-PTX cells, or a mild effect in 8505C-PTX cells is observed. Since these are resistant cells, treatment with low concentrations of paclitaxel obviously has no effect. However, as shown in Figure 2 panel C, combinations of paclitaxel and GSK2801 at different concentrations result in a strong decrease in cell viability, which suggests a partial resensitization of the cells to paclitaxel. The combination used for the following experiments is the one that showed the greatest reduction in viability (PTX 200 nM and GSK2801 20 µM). Given the reduction in cell viability after combined treatment with paclitaxel and GSK2801, we evaluated whether it could be due to apoptotic phenomena. For this purpose, cleaved PARP levels were assessed as a marker of apoptosis. Figure 2, panels D and E, shows the effects of simultaneous administration of paclitaxel and GSK2801 on cleaved PARP protein levels. As can be seen from the figure, a significant increase in apoptosis was observed in both cell lines following treatment.  After determining the treatment dose of GSK2801 alone and in combination with paclitaxel causing an important effect on cell viability, we investigated the effect of this molecule on the ability to form colonies in an anchorage-dependent way in the two cell lines. The two cell lines react differently when treated with GSK2801 alone. In 8505C-PTX cells, as shown in Figure 3 (panels A and C), the number of colonies formed after treatment with GSK2801 alone was greatly reduced compared with paclitaxel alone. When administered together with paclitaxel, GSK2801 had a greater effect in reducing colonies, which was significant compared with both single treatment and control. Regarding SW1736-PTX cells ( Figure 3 (panels B and D)), a significant reduction was observed only in combination treatment, whereas when treated with GSK2801 alone, the number of colonies was very similar to that obtained in the paclitaxel treatment situation.

Effects of GSK2801 on In Vitro Tumor Cell Aggressiveness
To investigate in more detail the effect of this drug on the aggressive behavior of cancer cells, we examined the ability of cells to form colonies in an anchorage-independent way. A well-established in vitro method to characterize this competence is soft agar assay [28]. As shown in Figure 4 (panels A and B), after treatment with 20 µM GSK2801 alone, we observed a strong significant reduction in the number of colonies only in treated SW1736-PTX cells (Figure 4

Effects of GSK2801 on In Vitro Tumor Cell Aggressiveness
To investigate in more detail the effect of this drug on the aggressive behavior of cancer cells, we examined the ability of cells to form colonies in an anchorage-independent way. A well-established in vitro method to characterize this competence is soft agar assay [28]. As shown in Figure 4 (panels A and B), after treatment with 20 μM GSK2801 alone, we observed a strong significant reduction in the number of colonies only in treated SW1736-PTX cells (panel B), while in combined treatment (200 nM PTX and 20 μM GSK2801), the number of colonies was significantly reduced in both 8505C-PTX and SW1736-PTXcells.
Another capacity related to tumor aggressiveness is the migration ability. We performed a scratch assay in order to explore this capacity of paclitaxel-resistant ATC cells in the presence or absence of treatment. The 2D wound healing assay is essentially based on the disruption of the cell monolayer at confluence, which generates a cell-free region, which can be re-covered by cells based on their ability to migrate [29]. Figure 4, panels C,D, and E, shows the analysis of the data obtained through this experiment in the time frame 0-6 (hours). In both cell lines, treatment with GSK2801 leads to acceleration of wound closure. In contrast, when cells were treated with both paclitaxel and GSK2801, their closure rate was strongly reduced compared with paclitaxel-only treatment. In panels (C,D), histograms represent the number of colonies. n = 3. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Another capacity related to tumor aggressiveness is the migration ability. We performed a scratch assay in order to explore this capacity of paclitaxel-resistant ATC cells in the presence or absence of treatment. The 2D wound healing assay is essentially based on the disruption of the cell monolayer at confluence, which generates a cell-free region, which can be re-covered by cells based on their ability to migrate [29]. Figure 4, panels C-E, shows the analysis of the data obtained through this experiment in the time frame 0-6 (hours). In both cell lines, treatment with GSK2801 leads to acceleration of wound closure. In contrast, when cells were treated with both paclitaxel and GSK2801, their closure rate was strongly reduced compared with paclitaxel-only treatment.

Effects of GSK2801 on Gene Expression in Paclitaxel-Resistant ATC Cells
To better understand the molecular mechanisms behind the biological effects exhibited by GSK2801 on paclitaxel-resistant ATC cells, a high-throughput RNA-sequencing analysis after GSK2801 treatment was performed. To detect direct effects of BAZ1B, BAZ2B, and BRD9 inhibition, GSK2801 or vehicle (DMSO) was administered at a concentration of 20 µM for an early time of 6 h. RNA extracted from four biological replicates for each experimental point was pulled and then subjected to high-throughput RNA-sequencing analysis, and for each cell line, samples treated with GSK2801 were compared against those that had received only DMSO. In SW1736-PTX cells, treatment with GSK2801 resulted in upregulation of 11495 line-specific genes and downregulation of 2518 line-specific genes. In 8505C-PTX cells, on the other hand, the same treatment resulted in increased expression of 2815 genes, while inducing reduced expression in 321 genes line-specific in both cases. Comparing commonly upregulated genes and commonly downregulated genes in the two cell lines, the administration of GSK2801 for 6 h increased the expression of 250 genes and reduced expression of 155 genes ( Figure 5, panel A). Overall, the effects of early treatment that inhibits BAZ2A, BAZ2B, and BRD9 appear to have an activating effect with an increase in gene expression in both lines, most evident in SW1736-PTX, probably because these cells when untreated (DMSO) showed lower gene expression levels ( Figure 5, panel B). Of particular interest was the analysis of commonly up-and downregulated genes in the two different cell lines. As shown in Figure 5 (panel C), the two pathways most affected by GSK2801 treatment were the cadherin signaling and the WNT signaling ones. Some genes that were part of these two pathways were upregulated in both cell lines, while others were downregulated. More specifically, the individual genes showing a change in expression in each pathway, or common to the two, are listed in Table 1.   Assuming greater cause-effect linearity between GSK2801 treatment and reduced expression levels rather than with increased gene expression, we focused on genes commonly downregulated in the two cell lines. After assessing the levels of gene expression reduction (Log2), their cellular function, basal expression levels, and grade of relevance in involvement in tumor, we chose MYCN as an important gene whose expression is reduced in both cell lines.

ChIP Analysis of the Effects of GSK2801 on the Binding of BRD9 to Its Targets
In order to evaluate the effects of GSK2801 on the interaction of BRD9 with its targets, a ChIP analysis was conducted using an antibody directed against BRD9. The fold enrichment of immunoprecipitation with anti-BRD9 antibody compared with nonspecific IgG for some already-known targets of BRD9 [30] was initially analyzed. As shown in Figure 6 (panel A), we demonstrated that the analyzed genes were confirmed to be BRD9 targets in our model. Treatment with GSK2801 displaced the binding of BRD9 to the targets tested, resulting in a significant decrease in enrichment (BRD9 vs. IgG) in most of them ( Figure 6, panel B). Among others, MYCN was shown to be a target of BRD9 and treatment with GSK2801 was able to disrupt this interaction.

ChIP Analysis of the Effects of GSK2801 on the Binding of BRD9 to Its Targets
In order to evaluate the effects of GSK2801 on the interaction of BRD9 with its targets, a ChIP analysis was conducted using an antibody directed against BRD9. The fold enrichment of immunoprecipitation with anti-BRD9 antibody compared with nonspecific IgG for some already-known targets of BRD9 [30] was initially analyzed. As shown in Figure  6 (panel A), we demonstrated that the analyzed genes were confirmed to be BRD9 targets in our model. Treatment with GSK2801 displaced the binding of BRD9 to the targets tested, resulting in a significant decrease in enrichment (BRD9 vs. IgG) in most of them ( Figure 6, panel B). Among others, MYCN was shown to be a target of BRD9 and treatment with GSK2801 was able to disrupt this interaction. Figure 6. Chromatin was isolated from SW1736-PTX and 8505C-PTX cell lines and immunoprecipitated with BRD9 or IgG antibodies. MYCN, E2F1, ELK4, MYO7, NFE2L1 DNAs were amplified using promoter-specific primers and analyzed by qPCR. All samples were run in triplicate. IgG-immunoprecipitate was arbitrarily set at 1.0 and the enrichment was expressed as relative expression Figure 6. Chromatin was isolated from SW1736-PTX and 8505C-PTX cell lines and immunoprecipitated with BRD9 or IgG antibodies. MYCN, E2F1, ELK4, MYO7, NFE2L1 DNAs were amplified using promoter-specific primers and analyzed by qPCR. All samples were run in triplicate. IgGimmunoprecipitate was arbitrarily set at 1.0 and the enrichment was expressed as relative expression value panel (A). SW1736-PTX and 8505C-PTX cells were treated with GSK2801 20 µM or with DMSO only (control). After chromatin was immunoprecipitated with BRD9 or IgG antibodies, MYCN, E2F1, ELK4, MYO7, NFE2L1 DNAs were amplified using promoter-specific primers and analyzed by qPCR panel (B). All samples were run in triplicate. Enrichment versus IgG of control (cells not treated with GSK2801) was arbitrarily set at 1 (control). n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Effects of GSK2801 on MYCN RNA and Protein Levels in SW1736-PTX and 8050C-PTX Cell Lines
After showing the MYCN expression reduction by treatment with GSK2801 ( Table 1) and considering that MYCN is a specific target of BRD9, the effects of such treatment on its protein levels were investigated. First, the decrease in MYCN RNA following treatment with GSK2801 20µM was reconfirmed, as shown in Figure 7 (panel A). Data obtained fit with what was observed by transcriptomics analysis. In addition, when evaluating MYCN protein expression by Western blot analysis, GSK2801 treatments induce a significant reduction in both cell lines (Figure 7, panel B and C).

Effects of GSK2801 on MYCN RNA and Protein Levels in SW1736-PTX and 8050C-PTX Cell Lines
After showing the MYCN expression reduction by treatment with GSK2801 (Table 1) and considering that MYCN is a specific target of BRD9, the effects of such treatment on its protein levels were investigated. First, the decrease in MYCN RNA following treatment with GSK2801 20μM was reconfirmed, as shown in Figure 7 (panel A). Data obtained fit with what was observed by transcriptomics analysis. In addition, when evaluating MYCN protein expression by Western blot analysis, GSK2801 treatments induce a significant reduction in both cell lines (Figure 7, panel B and C).

Effects of VPC-70619 on ATC Cell Viability and Colony Formation
Considering the relevance of MYCN in the synergistic mechanism of action of GSK2801 and paclitaxel, we evaluated whether a specific small molecule inhibitor of MYCN (VPC-70619) [31] would be able to alter the observed effects. First, the effect of MYCN inhibition was tested in terms of change in gene expression of its targets that were already described in previous paper [32]. Among the numerous targets described, a few were arbitrarily chosen whose expression would also result from RNA-seq in the cell lines under investigation.   Supplementary  Table S1.
Then, we investigated effects of VPC-70619 on the ability of tumor cells to form colonies in plate. For this reason, we performed a colony formation assay to evaluate whether there were differences between the number of colonies formed after treatment with paclitaxel alone, VPC-70619 alone, or with a combined treatment. The results are reported in Figure 9. As shown, the number of colonies formed after treatment with VPC-70619 decreases significantly in both cell lines. The effect of treatment with VPC-70619 alone was amplified when used in combination with paclitaxel in both cell lines.

Effects of VPC-70619 on In Vitro Tumor Cell Aggressiveness
Since the effects of GSK2801 on in vitro tumor aggressiveness, we repeated the analysis after MYCN inhibition by VPC-70619. VPC-70619 showed an inhibitory effect on soft agar colony formation already in single treatment but even more in combination treatment in both cell lines. The data are shown in Figure 10, panel A (8505C-PTX) and panel B (SW1736-PTX), and illustrate a similarity with those obtained in the previous experiment since again, the combined treatment has a greater inhibitory effect on colony-forming ability. Wound closure assay demonstrated that, in both cell lines, treatment with paclitaxel alone led to nearly complete wound closure (higher wound closure rate). Compared with this control situation, single treatment with VPC-70619 does not result in wound closure (Figure 10, panels C and D). This effect is more evident in the case of combined treatment; this means that VPC-70619 has a higher inhibitory effect on cell migratory capacity when used in combination with paclitaxel.

Effects of VPC-70619 on In Vitro Tumor Cell Aggressiveness
Since the effects of GSK2801 on in vitro tumor aggressiveness, we repeated the analysis after MYCN inhibition by VPC-70619. VPC-70619 showed an inhibitory effect on soft agar colony formation already in single treatment but even more in combination treatment in both cell lines. The data are shown in Figure 10, panel A (8505C-PTX) and panel B (SW1736-PTX), and illustrate a similarity with those obtained in the previous experiment since again, the combined treatment has a greater inhibitory effect on colony-forming ability. Wound closure assay demonstrated that, in both cell lines, treatment with paclitaxel alone led to nearly complete wound closure (higher wound closure rate). Compared with this control situation, single treatment with VPC-70619 does not result in wound closure ( Figure 10, panels C and D). This effect is more evident in the case of combined treatment; this means that VPC-70619 has a higher inhibitory effect on cell migratory capacity when used in combination with paclitaxel.  inhibitory effects of VPC-70619 on the ability to form colonies in an anchorage-independent way, which was studied by soft agar assay. The y-axis of each histogram shows the number of colonies formed for each condition (treatment with paclitaxel (PTX), VPC-70619 alone /VPC-70619) and combination (PTX + VPC-70619)). n = 3. Panels (C,D) show the change in cell migratory capacity after single and combined treatment, in both cell lines. Migratory capacity was evaluated by wound healing assay. The wound closure rate was calculated measuring the difference in wound width (WW) at time zero (t0) and after 6 h (t6). The formula used is as follows: Wound closure rate = (WWt0 − WWt6) treatment ÷ (WWt0 − WWt6) control. Panel (E): Photos showing wound margins (continuous black lines) at the beginning of migration (t0) and after 6 h (t6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Discussion
Whereas the combination of thyroid gland surgical excision and involved lymph nodes as well as irradiation with radioactive iodine (RAI) is effective for most thyroid tumors, this approach is not effective in benefiting the treatment of poorly differentiated thyroid carcinoma or ATC [33]. For this subclass of thyroid neoplasms showing such a high degree of malignancy, there are numerous therapeutic approaches that have been investigated in recent years [34][35][36]. However, identifying the right strategy to improve the life expectancy of ATC patients still remains a challenging task, partly because of the development of resistance to therapy. Our research group has been moving for years in the field of studying new therapeutic agents that can cope with the progression of ATC [37]. After experimenting with natural compounds, among others, we generated two cell lines resistant to paclitaxel treatment, derived from two anaplastic thyroid cancer cell lines (SW1736 and 8505C) [3]. Paclitaxel indeed, is a taxane still indicated in mono-or multi-therapy for those tumors in which the molecular signature does not suggest the use of personalized treatment with a tyrosine kinase inhibitor [4]. In our previous studies, we obtained interesting results regarding the gene expression profile of paclitaxel-resistant ATC cells compared with susceptible cells; moreover, we had already demonstrated the role of bromodomain and extra-terminal (BET) proteins and their inhibitors in this tumor type [3,38,39]. Therefore, we focused our attention on three genes coding for bromodomain-containing proteins (BAZ2A, BAZ2B, and BRD9) that were all expressed in paclitaxel-resistant SW1736 and 8505C cancer cell lines. First, we analyzed the expression of the three target genes demonstrating overexpression of BRD9 in paclitaxel-resistant cells, with no significant effect on the expression of BAZ2A and BAZ2B (Figure 1). Based on the increased expression of BRD9 and the importance of bromodomain-containing proteins in thyroid tumor development, tumor growth, and progression, we tested the efficacy of a specific inhibitor of these three proteins (GSK2801) [25]. In the literature, it has already been described that GSK2801 alone does not have particular effectiveness on triple-negative breast cancer cells, while its effectiveness increases sharply when used synergistically with another inhibitor [26]. GSK2801 showed no effect on cell viability of paclitaxel-sensitive SW1736 and 8505C cell lines, even when used at high doses (20 µM, 40 µM). When it was tested on paclitaxel-resistant ATC cells, consistently with what was observed in the literature, the administration of GSK2801 alone does not cause any particular effects, whereas in association with paclitaxel, important and statistically significant effects in terms of cell viability were observed ( Figure 2). This effect would be due to partial GSK2801-mediated resistant cell paclitaxel resensitization. Although further investigation into the mechanisms underlying the reduction in cell viability is needed, part of the phenomenon could be explained by the increase in cell death by apoptosis. Resistant paclitaxel ATC cells also showed a reduced ability to form plate colonies when treated with a combination of GSK2801 and paclitaxel (Figure 3). In addition to encouraging data on cell proliferation, the combined treatment showed great effectiveness in reducing the in vitro aggressiveness behavior, a feature evaluated by investigating the ability of cells to form colonies in soft agar or their ability to migrate. Again, GSK2801 when used alone did not appear to have significant effects, suggesting a possible reversion of resistance given the fact that it showed its effects only when used in combination with paclitaxel. In this study, we firstly demonstrated that the inhibition of BAZ2A, BAZ2B, and BRD9 protein activity by a specific inhibitor is able to strongly reduce the viability, to induce apoptosis, and to decrease clonogenic capacity of resistant paclitaxel cells when used in synergism. Furthermore, these effects also extended to in vitro tumor aggressiveness, which appeared significantly reduced in SW1736-PTX and 8505C-PTX cells when GSK2801 was co-administered with paclitaxel. Taken together, these results suggest that, since the action of GSK2801 appears significant only in synergy with paclitaxel, the mechanism of action may relate to partial resensitization of resistant cells. Therefore, it is possible to hypothesize that the bromodomain proteins inhibited by GSK2801 are no longer able to fulfill their function as regulators of gene transcription, thus altering the pattern of gene expression necessary to maintain resistance to paclitaxel treatment in these cells. In light of the marked biological effects of synergistic treatment, we then focused on the molecular mechanisms by which GSK2801 might alter the balance of ATC cells not sensitive to paclitaxel, making them susceptible to such treatment again. To this purpose, the effects of GSK2801 treatment on gene expression were investigated. To assess the most direct effects, an RNA-seq analysis was performed on RNA extracted from cells treated with GSK2801 since it is a competitive inhibitor that prevents its targets from interacting with proteins-DNA; its action does not have to go through, for example, downregulation of the expression of BAZ2A, BAZ2B, and BRD9 proteins. Since it immediately acts on the already available proteins, its effects can be presumed to be measurable as early as after initial treatment, when on the other hand, there is not yet an alteration in gene expression induced by cellular suffering secondary to treatment. In contrast to the number of genes with altered expression after treatment with GSK2801 in either cell line, the number of dysregulated genes commonly in SW1736-PTX and 8505C-PTX cells was more modest ( Figure 5). This is probably attributable to the fact that the two cell lines have different expression profiles, but the genes that respond to treatment are presumably few and specific. In light of this, the hypothesis that the effects of GSK2801 go through resensitization to paclitaxel of resistant cells finds additional support, as resistance to this taxane is an acquired characteristic shared by two different cell lines. Assuming a greater linearity of effect between treatment and downregulation of gene expression, genes in both cell lines underexpressed relative to controls were investigated. After evaluating the above genes by basal expression level, cell function, and relevance in tumorigenesis, the focus was on MYCN. MYC family members, MYC, MYCN, and MYCL, are transcription factors that regulate the expression of genes involved in normal development, cell growth, proliferation, metabolism, and survival. MYC dysregulation appeared to be tissue-specific tissue during tumorigenesis; MYCN was described to be aberrantly expressed in neuronal or neuroendocrine tumors, and more recently, in ovarian cancer, prostate cancer, and in a subset of breast cancer with poor prognosis [40][41][42]. The emergence in our analysis of MYCN as a likely most relevant target following treatment with GSK2801 would also be strongly supported by extensive literature on this topic, which shows that there is a strong correlation between overexpression of MYCN and response to treatment with BET inhibitors used alone or in combination [43,44]. MYCN turned out to be a direct target of BRD9 in this cell model (Figure 6), and inhibition of MYCNtarget binding acted by GSK2801 determined a marked and significant reduction in MYCN protein levels in SW1736-PTX and 8505C-PTX cells (Figure 7). Based on the hypothesis that MYCN was one of the main actors downstream of the biological effects of GSK2801, a specific MYCN inhibitor was tested to assess whether it could mimic the effects of a MYCN downregulation. SW1736-PTX and 8505C-PTX cells were treated with VPC-70619, a small molecule inhibitory MYCN [31], at a concentration at which, when used alone, it was unable to determine effects on cell viability (2 µM). VPC-70619 also demonstrated its specificity of direct action against MYCN in paclitaxel-resistant SW1736 and 8505C cells, as was shown by the change in expression levels of some of its targets (Figure 8). MYCN itself, MDM2, and CRABP2 were upregulated after MYCN inhibition, while TGM2 and INHBA were downregulated, in agreement with what was already observed by Bell et al. [32]. Similar to that observed in co-treatment with GSK2801 also in the case of simultaneous administration of VPC-70619 and paclitaxel, a strong reduction in cell viability was observed, at concentrations at which neither drug had an effect individually (Figure 8). The effective dose of VPC-70619, when used in cotreatment with paclitaxel, was 10 times lower than that of GSK2801 (2 µM vs. 20 µM). This could be due to the fact that BRD9 acts as a modulator of gene expression of a plethora of genes; its inhibition therefore induces fewer direct effects than specific suppression of MYCN. Even in this case, co-treatment of VPC-70619 and paclitaxel showed inhibition of plate colony formation and reduction of tumor aggression, as demonstrated by soft agar assay and wound healing assay. In conclusion, inhibition of MYCN activity by VPC-70619 showed significant biological effects on paclitaxel-resistant ATC cell lines when used together with paclitaxel. This suggests that the lack of functionality of MYCN may result in partial resensitization of the cells examined and, ultimately, that a substantial part of the effect of GSK2801 comes through inhibition of MYCN expression. This preliminary study opens a way to understanding the mechanisms of how BET proteins exert their role in anaplastic thyroid cancer and how their inhibition may represent a therapeutic target for the treatment of a type of tumor which is to date still hardly curable. In addition, these results offer a molecular explanation of the observed effects by applying MYCN to high-profile targets in this type of mechanism of action. As far as we know, this is the first study in which the effects of BAZ2A, BAZ2B, BRD9, and MYCN inhibition in paclitaxel-resistant anaplastic thyroid carcinoma cell lines, are examined, providing a starting point for further and more in-depth studies that can fill the gap of effective treatments for this subcategory of thyroid tumors.
Cell viability In order to test cell viability, the MTT assay was used, as previously described [34]. SW1736-PTX and 8505C-PTX cells were seeded in 96-well plates (4 × 10 3 cells/well). The following day, cells were treated with PTX or GSK2801 at different concentrations and different combinations. After 72 h of incubation, 4 mg/mL MTT (Sigma-Aldrich, Darmstadt, Germany; Merck KGaA, Darmstadt, Germany) was added to the cell medium and cells were cultured for a further 4 h in the incubator. The supernatant was removed, 100 µL/well DMSO was added, and the absorbance at 570 nm was measured. The same experiment was performed with paclitaxel or VPC-70619 treatment, following the same protocol. All experiments were performed as six technical repeats and cell viability is expressed as the fold-change relative to the control. EC50 was determined from the dose-response curves using the percentage of cell viability.
Colony formation assay The clonogenic activity of the paclitaxel-resistant ATC cell lines was evaluated by colony formation assay. Briefly, SW1736-PTX and 8505C-PTX cells were treated with paclitaxel, GSK2801, or in combination and then the cells were seeded in 10-cm plates at a density of 1000 cells/plate. Adherent colonies were fixed using methanol and then stained with crystal violet colorimetric dye, which allows for visual quantification of the number of colonies that expanded. The same experiment was performed with paclitaxel or VPC-70619 treatment, following the same protocol.
Soft agar assay The clonogenic ability of the SW1736-PTX and 8505C-PTX cells was studied using a soft agar assay. After 48 h of treatment, cells were collected and 20,000 cells were suspended in complete medium containing 0.25% agarose (PanReac AppliChem ITW Reagents, Darmstadt, Germany), then seeded to the top of a 1% agarose complete medium layer in 6 cm plates. The colonies were counted in four different fields under a Leica DMI-600B inverted microscope (Leica Microsystems Ltd., Wetzlar, Germany). The same experiment was performed with paclitaxel or VPC-70619 treatment, following the same protocol. Data are representative of three independent experiments.
Wound healing assay The migration capability of the cells was evaluated by wound healing assay. The cells were seeded into 6-well plates (500,000 cells/well) and the day after they were treated with paclitaxel, GSK2801, or with a combined treatment, or in the second